Abstract Thin filament-linked actin-binding proteins, troponin and tropomyosin, control actomyosin-based muscle contraction in cardiac and skeletal muscles. To elucidate mechanisms of muscle thin filament function, it is crucial to determine the changing structural interactions of these regulatory proteins at a fundamental molecular level. It follows that disease-related myofibrillar protein mutants can perturb muscle on-off switching by causing an imbalance in troponin-tropomyosin interactions on actin which, in turn, destabilizes relaxed or activated states and transitions between them. Such imbalances, either intrinsic to troponin-tropomyosin regulation itself or caused indirectly by the effects of actin, myosin or myosin-binding protein-C, are expected to modify muscles? cooperative activation and relaxation as well as allosteric communication pathways between thin filament components. In the current work, we will use state-of-the-art cryo-electron microscopy, coupled with image analysis and 3D recon- struction, to establish the macromolecular structure of native troponin and tropomyosin on thin filament actin. We will combine this approach with Molecular Dynamics simulations as well as other computational tools to define transitions between thin filament regulatory states in response to Ca2+ effects on troponin and myosin-binding to actin. In order to understand how mutations can initiate aberrant physiology leading to pathology, we will compare structural interactions and transitions that occur normally in thin filaments with those in filaments containing mutant proteins linked to myopathies. To accomplish our goals: (1) we will generate high-resolution near-atomic level models of troponin-tropomyosin on native and reconstituted thin filaments by cryo-EM (Specific Aim 1); (2) we will reveal the transition pathways between thin filament states in energy landscapes and by targeted Molecular Dynamics, accounting for the underlying stereochemistry and material properties of tropomyosin and troponin required for the cooperative transitions (Specific Aim 2). (3) Finally, aiming to develop tools to counteract regulatory imbalances, we will manipulate cooperative, regulatory pathways using small molecules trapped in pockets present in overlap connections between successive tropomyosin molecules along actin filaments (Specific Aim 3). A coupling of the understanding of near atomic level mutational ?insults? that perturb muscle control mechanisms alongside prospects of reversing early-stage alterations in physiological function has broad biomedical significance. Here, the design of well-targeted small molecule compounds to manipulate muscle regulation has the potential to translate into future therapeutic platforms. Our work on striated muscle thin filaments pays particular attention to the function of troponin-tropomyosin, proteins at the hub of cooperative regulation of skeletal and cardiac muscle contraction. Moreover, because smooth muscle contractility also is modulated via tropomyosin and other actin- binding proteins, our work will also foster better understanding of thin filament control of vascular tone and pulmonary airway resistance. Furthermore, the fact that non-muscle tropomyosins participate in cytoskeletal remodeling in virtually all cells underscores the broad significance of the proposed work.